ADDIS ABABA SCIENCE AND TECHNOLOGY UNIVERSITY

COLLEGE OF BIOLOGICAL AND CHEMICAL ENGINEERIN

DEPARTMENT OF BIOTECHNOLOG

ADVANCED MICROBIA BIOTECHNOLOGY

Prepared by Genet Girma

ID gsr 295/14

Submitted to Dr Ebrahim mama

Submission date 15 /06 / 2022

Table of content
Abstract

Strain improvement/metabolic engineering of E.coli for the formation of value added product is
essential methods to produce product with high yield and quality to address the demands. Lactic
acid is the most common value added product with different application. D-lactate and l-lactate are
the chemical isomers of lactic acid. In this term paper lactic acid production is mainly focused by
improving the E.coli strain. The methods for production of lactic acid , application, types of
fermentation, and the substrates that used in the production of lactic acid industrially is also
discussed.
 1. Introduction

Lactic acid bacteria (LAB) are a general term for a class of bacteria that use the metabolism of
carbohydrates in the external environment to produce lactic acid. Lactic acid bacteria are widely
distributed in nature and exist in a variety of habitats. Extremely rich in biodiversity, they are closely
related to human production and life and have important social and economic value.[Tian, F. (2019).
Introduction. In: Chen, W. (eds) Lactic Acid Bacteria. Springer,
Singapore.https://doi.org/10.1007/978-981-13-7832-4_1]. For humans, they are vital biological
resources. The use of lactic acid bacteria by humans dates back thousands of years.Lactic acid
bacteria (LAB) are responsible for a great diversification in the flavor and texture of food products
due to their fermentation of food raw materials. However, in some circumstances they can be
responsible for food spoilage[König,]. It can be traced back to more than 10,000 years ago, according
to trustworthy archaeological data. LAB was amongst the very first bacteria studied. In 1873, Joseph
Lister isolated the first bacterial pure culture which he called Bacterium lactis. This lactic acid
bacterium is now called Lactococcuslactis and is used for fermenting milk to produce hundreds of
different dairy products. Early work on LAB was mainly concentrated on those associated with dairy
products and, with time, many commercial starter cultures were developed.[König, H., &Fröhlich, J.
(2017).Lactic acid bacteria.In Biology of Microorganisms on Grapes, in Must and in Wine (pp. 3-41).
Springer, Cham.]Lactic acid bacteria have made significant contributions to the formation and
practice of human society over the course of history. The science and technology of lactic acid
bacteria had tremendous development during the late nineteenth and early twentieth centuries,
along with the rapid development of biological science, microbiology, and other related areas.On the
one hand, lactic acid fermentation is used as a model metabolic technique in basic research. Lactic
acid bacteria are significant theoretical research organisms in basic biological disciplines such as
microbiology, biochemistry, genetics, and molecular biology. Lactic acid bacteria, on the other hand,
offer a wide range of practical applications in industrial biomanufacturing, food production and
processing, high-efficiency agriculture, high-efficiency animal breeding, medical health, and other
key human-related disciplines.The current consumption of lactic acid is growing considerably, due to
its use as a monomer for the synthesis of biodegradable polymer, poly lactic acid (PLA). Hence, it is
necessary to increase the yield of lactic acid product through different production improvement
strategies such as metabolic engineering. Industrially, lactic acid can be produced by chemical
synthesis or by fermentation.[Komesu, A., de Oliveira, J. A. R., da Silva Martins, L. H., Maciel, M. R.
W., &MacielFilho, R. (2017). Lactic acid production to purification: a review. BioResources, 12(2),
4364-4383.]

Metabolic engineering is a potent biotechnological method that is increasingly being used in the
construction of microbial strains for improved lactic acid productivity, cheaper costs, and reduced
pollution, among other applications. The focus of metabolic pathway engineering has been on
improving lactic acid fermentation parameters, increasing producing organisms' acid tolerance and
their ability to use a wide range of substrates, including fermentable biomass-derived sugars. Lactic
acid bacteria have been the focus of recent studies because they produce high yields and have a
small genome size that makes genetic manipulation easier[Upadhyaya, B. P., DeVeaux, L. C., &
Christopher, L. P. (2014). Metabolic engineering as a tool for enhanced lactic acid production.Trends
in biotechnology, 32(12), 637-644.].
Ideal characteristics of strain

A strain to be selected for fermentation process should satisfy the following criteria- it should be
able to produce the desired product and produce the product at large scale, it should be a well-
known organism, it should be able to grow faster, it should be safe to handle, it should grow in
minimal to moderate growth media, it should have optimum growth temperature considerably
above 400c, reduces the cooling costs and will be beneficial for isolation procedures at large scale
fermentation processes, it should be genetically stable and simple to understand, it should be easier
to manipulate it at genetic level, product recovery should be easy from the culture
[https://www.slideshare.net/jeevaraj9/strain-improvement-techniques].

Methods for strain improvement

["https://www.biologydiscussion.com/biotechnology/bioprocess-technology/genetic-improvement-
of-strains-features-and-methods/10108" /]

Mutagenesis

Mutation is a term used to describe any alteration in a gene's DNA. As a result, mutations cause
structural changes in the genome. Mutations can be either spontaneous (naturally occurring) or
caused by mutagenic agents. Because spontaneous mutations occur infrequently, they are usually
unsuitable for industrial use. Mutations can be caused by mutagenic factors like ultraviolet radiation
or chemicals (nitrous oxide, nitrosoguanidine, and hydroxylamine). For strain improvement, site-
directed mutagenesis is also significant.

Genetic Recombination:

Genetic recombination also important strain improvement methods that can be made by combining
genetic information from two genotypes, by a process called genetic recombination. The
recombination can be brought out by transformation, transduction, conjugation and protoplast
fusion.

2. Lactic acid

LAB produces ATP by glucose fermentation and phosphorylation at the substrate level. The glycolytic
pathway (Embden-Meyerhof pathway) (Fig. 1), which produces lactic acid as the main end product
(homofermentative metabolism), and the phosphoketolase pathway (Fig. 2), which produces other
end products such as acetic acid, propionic acid, CO2, ethanol, and others in addition to lactic acid
(heterofermentative metabolism) [Papagianni M. (2012). Metabolic engineering of lactic acid
bacteria for the production of industrially important compounds.Computational and structural
biotechnology journal, 3, e201210003. https://doi.org/10.5936/csbj.201210003]. In substrates with
rapidly metabolized sugars, L. lactis shows homofermentative metabolism, with more than 90% of
the metabolized sugar being transformed to lactic acid.Lactic acid bacteria are bacteria that produce
lactic acid as a significant metabolic end product of carbohydrate fermentation[König,].. They are
commonly found in decomposing plants and milk products (LAB). Production of lactic acid has linked
LAB with food fermentations, as acidification inhibits the growth of spoilage agents.
Proteinaceousbacteriocins are produced by several LAB strains and provide an additional hurdle for
spoilage and pathogenic microorganisms. Furthermore, lactic acid and other metabolic products
contribute to the organoleptic and textural profile of a food item. The industrial importance of the
LAB is further evidenced by their generally recognized as safe (GRAS) status, due to their ubiquitous
appearance in food and their contribution to the healthy microbiota of animal and human mucosal
surfaces[//en.m.wikipedia.org/wiki/Lactic_acid_bacteria"/>].Improved nutritional value of food,
control of intestinal infections, improved digestion of lactose, control of some types of cancer, and
control of serum cholesterol levels are some of the use of lactic acid[Gilliland SE. Health and
nutritional benefits from lactic acid bacteria. FEMS Microbiol Rev. 1990 Sep;7(1-2):175-88. doi:
10.1111/j.1574-6968.1990.tb04887.x. PMID: 2271223.].Chemically, lactic acid occurs as two optical
isomers, a dextro and a levo form; only the levo form takes part in animal metabolism. The lactic
acid of commerce is usually an optically inactive racemic mixture of the two isomers [Chahal, S. P., &
Starr, J. N. (2000). Lactic acid.Ullmann'sencyclopedia of industrial chemistry.].Lactic acid bacteria can
tolerate low pH, high concentrations of salt, and heat treatments. Food-grade lactic acid bacteria
that are used as preservatives must be able to survive freezing, drying, and storage conditions. The
physical and chemical characteristics, the antimicrobial mechanisms and the stability of the
compounds when added to a food product must be determined. In fact, the concentration of acids,
salts, spices, chemical preservatives, and bacteriocins will affect the growth of the lactic acid
bacteria.

Glycolysis (Embden-Meyerhof pathway).

Phosphoketolase pathway

3. Common methods for lactic acid production

Chemical synthesis and fermentation is the common method by which lactic acid can be produced
industrially. Fermentation through microbes has potential advantages over that of chemical
synthesis in that pure lactic acid can be attained whereas, chemical synthesis of lactic acid always
give a raceme mixture. Lactic acid bacteria are the most common bacteria that produce lactic acid,
and among this Lactobacillus spp. have shown promising fermentation abilities. The usage of Bacillus
spp. produced promising results in terms of lowering fermentative expenses.
Corynebacteriumglutamicum and E. coli, for example, were able to produce large amounts of lactic
acid after undergoing genetic engineering [Abedi, E., &Hashemi, S. (2020). Lactic acid production -
producing microorganisms and substrates sources-state of art.Heliyon, 6(10),
e04974.https://doi.org/10.1016/j.heliyon.2020.e04974].Most of the world’s commercial lactic
acid is prepared by fermentation of carbohydrates by bacteria using homolactic microbes
such a variety of modified or optimized strains the genus Lactobacilli, which specially
produce lactic acid. Commercially pure lactic acid can be synthesized by microbial
fermentations.Although racemic lactic acid is always made by chemical synthesis from
petrochemical resources, optically pure L(+)- or D(-)-lactic acid can be made by microbial
fermentation of renewable resources with the right microbe. One kind of optically pure lactic
acid is preferred over the other depending on the purpose. Microbial fermentation also has a
number of advantages, including the use of low-cost renewable substrates, low-temperature
production, and low-energy usage. Because of these benefits, the manufacturing procedure is
the one that is most commonly employed [Komesu, A., de Oliveira, J. A. R., da Silva
Martins, L. H., Maciel, M. R. W., &MacielFilho, R. (2017). Lactic acid production to
purification: a review. BioResources, 12(2), 4364-4383.].

Figure 1Overview of the two manufacturing methods of lactic acid, chemical synthesis and microbial fermentation
(komesu et al .2017)

4. Substrate for LA production

Cheap raw ingredients are required to manufacture large amounts of lactic acid through
fermentation at a low cost. Apart from being inexpensive, raw materials should also have
features such as high yield, minimal or no byproduct generation, high productivity, and low
contamination, such that minimal pretreatment is required. The use of refined ingredients,
such as carbohydrates, can significantly reduce product purification costs, but it is not cost-
effective because it results in high production costs. Whey and molasses, which are
industrial wastes, are commonly used as substrates for lactic acid production. Whey contains
lactose, protein, fat and mineral salts as it are a by-product of dairy industry.
Microorganisms such as Lactobacillus helveticus and Lactobacillus casei are used in
production of lactic acid from whey[Krishna, B. S., Nikhilesh, G., Tarun, B., Saibaba, N.,
&Gopinadh, R. (2018). Industrial production of lactic acid and its applications. International
Journal of Biotech Research, 1(1), 42-54.]
5. LA producing microorganism
5.1. Fungi
Though lactic acid bacteria execute the majority of the lactic acid generation activities, some
fungal species, such as Rhizopus, can convert starch to L(+) lactic acid via their amylolytic
enzyme activity. Other benefits of fungal fermentation over bacterial fermentation include a
lower cost downstream process, lower nutrient requirements, and the creation of fungal
biomass, which is a valuable by-product. Because fungal fermentation uses a chemically
specified substrate, product purification is simple. This is a significant benefit in the food
sector[Krishna.
5.2. Yeasts
All the fermentation processes require abundant amount of nutrient supply. In many of the
fermentation processes, not only lactic acid, yeast is used as the key nutrient source. The
major advantages of using yeast as nutrient source include their tolerance against low pH
(1.5), which prevents the regeneration of precipitated calcium lactate, thereby reducing the
cost of neutralization by neutralizing agents such as calcium carbonate, and their ability to
grow in mineral media. Fermentation with wild-type yeast as a nutrition source produces a
low production of lactic acid. Genetically modified yeasts capable of producing significant
yields of lactic acid have been generated and are being used successfully since the advent of
genetic engineering.The main drawback of using yeast as a nutrient source is that it leads to
increase in production costs.
5.3. Bacteria
Lactic acid bacteria (LAB), Escherichia coli, Corynebacteriumglutamicum, and Bacillus strains
are the four primary groups of lactic acid producing bacteria. Lactic acid bacteria are the
most often used of these. It is critical to select the right strain because it affects
characteristics like as yield, productivity, purity, and nutritional requirements. One of the
key reasons for usage of lactic acid bacteria in industries is because it does not have any
adverse health effects. The properties such as high acid tolerance and the ability to be
engineered for selective production of D-or L-lactic acid make lactic acid bacteria
commercially useful [Krishna.

There are two types of LAB: homofermentative bacteria and heterofermentative bacteria.
Sugars are virtually completely converted to lactic acid by homofermentative LAB. Due to
the ability of production qualitatively and quantitatively homofermentative LAB used in
production of lactic acid for commercial purpose. The heterofermentative bacteria, on the
other hand, create not only lactic acid but also ethanol/acetic acid and carbon dioxide. In
practice, a test for glucose-induced gas generation will identify the two groups. There were
also differences in the rate of development at various temperatures, the pH of the media,
and salt chloride tolerance. Growth is generally assessed at temperatures of 18 and 45
degrees Celsius, sodium chloride concentrations of 6.5 and 18 percent, and pH levels of 4.4
and 9.3. Finally, various isomeric forms of lactic acid (L-lactic acid or D-lactic acid) might be
used to differentiate across genera[Ouwehand, A. (1993). Lactic acid bacteria (pp. 199-225).
S. Salminen,& A. Von Wright (Eds.). New York, USA: Dekker.] .
 6. Fermentation method for lactic acid production
Fermentation of lactic acid, like any other fermentation process, is dependent on factors
such as raw materials used, nutrients present in media, and the microorganisms used. Three
different methods of fermentation are practiced, namely, Batch fermentation, Fed-batch
fermentation, and Continuous fermentation.
6.1. Batch fermentation
In batch fermentation all essential ingredients, such as carbon source, nitrogen source, and
other components, are added prior to the start of the fermentation process. It is the most
often used fermentation method since it is straightforward to carry out. Batch fermentation
has the advantage of preventing contamination to a greater extent than other procedures
since it is a closed system that produces large quantities of lactic acid. The drawbacks of
batch fermentation include low productivity due to substrate inhibition or product
inhibition, and as the amount of nutrients provided is limited, low cell concentrations are
obtained.
6.2. Fed-batch fermentation

One way of keeping nutrients from becoming a limiting factor is to constantly supply them
during cultivation. This is called a fed-batch process, which is a partly open system. The
advantage of feeding during cultivation is that it allows to overall achieve higher product
quantities overall. Under specific growth conditions, the microorganisms and/or cells
constantly double and therefore follow an exponential growth curve. This is why the feed
rate should increase exponentially as well. Generally, the substrate is pumped from the
supply bottle into the culture vessel through a silicone tube. The user can either manually
set the feed at any time (linear, exponential, pulse-wise), or add nutrients when specific
conditions are met, such as when a certain biomass concentration is reached or when a
nutrient is depleted[Khamseh, A. A. G., &Miccio, M. (2012). Comparison of batch, fed-batch
and continuous well-mixed reactors for enzymatic hydrolysis of orange peel wastes. Process
Biochemistry, 47(11), 1588-1594.]

.
6.3. Continuous fermentation

Continuous fermentation is an open system in which the solution is continuously added to

and discharged from the system. Microorganisms and sterile nutrient solution are
continually fed to the bioreactor, and the nutrient solution and microorganisms are
transformed in the system in equal amounts[Maxon, W. D. (1955). Continuous fermentation:
A discussion of its principles and applications. Applied microbiology, 3(2), 110-122.].Allows
the maximum productivity, Time for cleaning, sterilisation and handling of the vessel are all
reduced and provides a steady state for metabolic studies when many elements sum to zero
this some of its advantages.Difficult to keep a constant population density over prolonged
periods and increased risk of contamination and/or genetic changes are drawbacks with
continuous fermentation.

7. Metabolic engineering of lactic acid production

Lactic acid is a very important industrial product. To enhance lactic acid production, a variety
of traditional and modern biotechnology techniques have been tested. Lactic acid bacteria,
yeast, and fungal systems have been genetically modified to boost lactic acid production.
With the advent of biotechnology and the awareness of lactic acid's industrial potential,
efforts have been concentrated on using biotechnological methods to produce lactic acid
bacteria (LAB) and other systems for lactic acid generation[Singh, S. K., Ahmed, S. U.,
&Pandey, A. (2006). Metabolic engineering approaches for lactic acid production. Process
Biochemistry, 41(5), 991-1000.].Metabolic engineering is a potent biotechnological method
that is increasingly being used in the construction of microbial strains for improved lactic
acid productivity, cheaper costs, and reduced pollution, among other applications. The focus
of metabolic pathway engineering has been on improving lactic acid fermentation
parameters, increasing producing organisms' acid tolerance and their ability to use a wide
range of substrates, including fermentable biomass-derived sugars. Lactic acid bacteria have
been the focus of recent studies because they produce high yields and have a small genome
size that makes genetic manipulation easier[Upadhyaya, B. P., DeVeaux, L. C., &
Christopher, L. P. (2014). Metabolic engineering as a tool for enhanced lactic acid
production. Trends in biotechnology, 32(12), 637-644.]. Rapidly growing demand for
polylactic acid has led to a rapid increase in lactic acid demand worldwide. According to the
explanation of the Computational and Biotechnology Journal Review of 2012, the target for
metabolic engineering aiming at increased lactic acid production levels lies in the area of
sugar utilization and the subsequent glycolytic and lactate fluxes. Regulation of glycolysis
and the shift between different fermentation modes have been extensively studied with L.
lactis. The role of phosphofructokinase (PFK) on the glycolytic flux in L. lactis, shows that the
particular enzyme plays an important role as both the glycolytic and lactate fluxes were
decreased proportionally by a two-fold reduction of PFK activity.However, as lactic acid
bacteria have complex nutritional requirements and very low growth rates; genetic
engineering of E.coli is the promising approach to enhance the productivity of lactic acid
industrially.
7.1. Metabolic engineering of E.coli

Escherichia coli have many advantageous characteristics as a production host, such as rapid
growth under aerobic and anaerobic conditions and simple nutritional requirements.
Moreover, well-established protocols for genetic manipulation and a large physiological
knowledge base should enable the development of E. coli as a host for production of
optically pure D- orL-lactate by metabolic engineering [.Chang, D. E., Jung, H. C., Rhee, J.
S., & Pan, J. G. (1999). Homofermentative production of d-orl-lactate in metabolically
engineered Escherichia coli RR1. Applied and environmental microbiology, 65(4), 1384-
1389.]
Here we are going to investigate metabolic engineering pathway of E.coli for production of
lactic acid. A pta mutant of E. coli RR1 that was deficient in the phosphotransacetylase of the
Pta-AckA pathway was shown to metabolize glucose to D-lactate and produce a small
amount of succinate by-product under anaerobic conditions, according to the researchers. A
further mutation in ppc caused the mutant to manufacture D-lactate in the same way as a
homofermentative lactic acid bacterium does. More than 62.2 g of D-lactate per liter was
produced in 60 hours when the ptappcdouble mutant was grown to higher biomass
concentrations under aerobic conditions before shifting to the anaerobic phase of D-lactate
synthesis. The volumetric productivity was 1.04 g/liter/h.To examine whether the blocked
acetate flux could be reoriented to a nonindigenousL-lactate pathway, an L-lactate
dehydrogenase gene from Lactobacillus casei was introduced into aptaldhA strain which
lacked phosphotransacetylase andD-lactate dehydrogenase. This recombinant strain was
able to metabolize glucose to L-lactate as the major fermentation product, and up to 45 g of
L-lactate per liter was produced in 67 h. These results demonstrate that the central
fermentation metabolism of E. coli can be reoriented to the production of D-lactate, an
indigenous fermentation product, or to the production of L-lactate, a nonindigenous
fermentation product. E. coli, a facultative anaerobe, performs mixed-acid fermentation of
glucose, yielding formate (or CO2 and H2), acetate, D-lactate, succinate, and ethanol (4) as
the main products. The entire fermentation balance or by-product pattern is considerably
affected by mutations in a given fermentation pathway(s). Pta mutants, which are unable to
produce phosphotransacetylase (Pta), have been found to be unable to grow or synthesis
acetate anaerobically on glucose minimum medium. Similarly, an adhmutant lacking alcohol
dehydrogenase (ADH) was unable to grow anaerobically on glucose. Lactate fermentation
allowed adhpta double mutants to thrive anaerobically on glucose. Therefore, the acetate
pathway appears to be one of the target pathways which can be manipulated to redirect the
fermentation metabolic flux of E. coli to lactate production.
So here, using ptamutants defective in the Pta-AckA acetate production pathway, we investigated
redirection of the metabolic flux from the acetate pathway to D- orL-lactate production. During
anaerobic cultivation of theE.coli RR1 ptappc mutant, fermentative metabolism was redirected to D-
lactate production for recycling of NADH produced by glycolysis. When L-LDH fromLactobacillus casei
was introduced into a ptaldhA mutant lacking enzymes leading to the production of acetate and D-
lactate, optically pure L-lactate was produced as the major fermentation product. The lactate
productivity was increased by growing the cells first under aerobic conditions before shifting them to
anaerobic conditions, which are favorable for the production of lactate.Dong-Eun Chang et.,al1999
investigated that an E. coli RR1 pta mutant was used as the host for production of optically pure d-
or l-lactic acid. A ptappc mutant was able to metabolize glucose exclusively to d-lactate under
anaerobic conditions, and a ptaldhA mutant harboring the l-LDH gene from L. casei produced
optically pure l-lactate as the major fermentation product.
Conversion of sucrose to lactic acid
An E. coli engineered to accumulate D-lactic acid from sucrose was constructed by a
knockout in the sucrose repressor gene (cscR), leading to 85 g D-lactic acid l−1 in 84 h with a
yield of 0.85 g/g (Wang et al. 2012). A strain engineered to accumulate L-lactic acid, also
evolved for improved growth on sucrose, resulted in the generation of 97 g L-lactic acid l−1
with a yield of 0.9 g/g in a defined medium containing sucrose (Wang et al. 2013). This strain
also converts sugarcane molasses and corn steep liquor to 75 g L-lactic acid l−1 with a yield
of 0.85 g/g (Wang et al. 2013).[Eiteman, M. A., &Ramalingam, S. (2015). Microbial
production of lactic acid. Biotechnology letters, 37(5), 955-972.]

Production d-lactate in genetically engineered E.coli

D-lactate is a key chiral intermediate utilized in the manufacture of a variety of medicinal,

insecticide, and chemical products. Polylactic acid (PLA), a renewable and biodegradable
polymer, is the most common application of D-lactate. Both the physical properties and the
rate of biodegradation of PLA are linked to the isomeric form, which has led to a greater
demand for a method of producing chirally and chemically pure D-lactate with high yields
and productivities[Zhou, L., Cui, W. J., Liu, Z. M., & Zhou, Z. M. (2016). Metabolic engineering
strategies for D‐lactate over production in Escherichia coli.Journal of Chemical Technology &
Biotechnology, 91(3), 576-584.].Due to the imminent scarcity of fossil chemical sources,
industrial-scale fermentation may become increasingly relevant in the manufacture of D-
lactate. Lactic acid bacteria are extensively used in the production of lactate in industry
(Sporolactobacillus sp. CASD produced D-lactate at a rate of 226 g L1). However, a medium
with high nutrition is required, which raises the cost of the bulk product, PLA, and impedes
not just lactate separation but also purification. With fewer metabolic engineering options
used in lactic acid bacteria, the fermentation cycle is significantly prolonged, and genetic
manipulation is much more challenging. Recombinant Corynebacteriumglutamicum could
also produce high level of D-lactate (the highest level reached 195 g L−1).4, 5 However,
additional nutrition is required, and the genetic manipulation of C. glutamicum becomes
difficult with higher accumulation of by-products. Bacillus coagulans, fungi and yeasts have
also been engineered for D-lactate production with significantly lower D-lactate titer (the
 highest level reached 89 g L−1), which is not appropriate for industrial production. In
contrast, E. coli has the advantages of producing optical D-lactate with rapid growth, simple
nutrition requirement, and easy genetic manipulation and has been successfully used for
industrial D-lactate production. E. coli has been studied extensively on a fundamental and
applied level and is the predominant host microorganism for industrial production of
industrially important biochemicals. Therefore; E. coli could be a good example to reveal the
metabolic engineering strategies applied for D-lactate production, which would be also
meaningful for the overproduction of other chemical products in E. coli.

In E. coli, D-lactate is the dehydrogenation product of pyruvate, which is an important intermediate

of sugar metabolism (Fig. 1). Consequently, the activity of fermentative lactate dehydrogenase
(LDH), the degradation rate of the carbon resource, and the cellular redox state all directly control
the synthesis of D-lactate. The flux through the competing metabolic routes decreases the yield and
chemical purity of D-lactate; the energy metabolism and the cellular components in the remote
metabolic routes affect the cell viability and indirectly regulate the production of D-lactate.
Accordingly, a metabolic engineering strategy that includes conventional deletion of competing
pathways, regulation of the redox state, coordination of the lactate production rate with cell
viability, and the introduction and/or amplification of various substrate metabolism pathways is
required to regulate these reactions to overproduce D-lactate with a high titer, yield and
productivity in a process that reduces the production cost and that precisely controls the
fermentation process in E. coli.

General metabolic pathway for the production of D-lactate from various substrates by E. coli strains. Genes and
enzymes: cscB sucrose permease (anion symport), cscA sucrose hydrolase
(invertase), cscK fructokinase, glk glucokinase, pts posphotransferase system
(PTS), pfkAB phosphofructokinase, fba fructose-1,6-bisphosphate aldolase, gatC galactitol-specific enzyme IIC
component of PTS, xylFGH xylose ABC transporter, glpK glycerol kinase, glpD aerobic glycerol-3-phosphate
dehydrogenase, gldA glycerol dehydrogenase, dhaKLM dihydroxyacetone kinase, ldhA fermentative D-lactate
dehydrogenase. The abbreviated metabolic intermediate are: PEP phosphoenolpyruvate and QH 2 reduced
quinines. The dotted arrow shows the same type of metabolic chemical. A closed circle indicates that the gene was
deleted.

Deletion of competing routes for lactate dehydrogenase

In anaerobic conditions, wild type E. coli performs mixed-acid fermentation to maintain a

redox balance and to optimize cellular growth through the anaerobic conversion of glucose
to mixtures of D-lactate, acetate, succinate, formate, ethanol, carbon dioxide and
hydrogen,resulting in a limited D-lactate yield of 40%. To improve the yield and chemical
purity of D-lactate, conventional metabolic engineering strategies deleting the by-product
synthesis routes with D-lactate accumulation.
Effects of single gene deletion
The effects of the competing routes have primarily been identified by single gene deletions
in wild type E. coli. The most effective gene deletions in improving lactate production are
found to be the deletions of pflA/pflB (pyruvate formatelyase, but not pflC and pflD), ackA
(acetate kinase) and pta (phosphotransacetylase). Pyruvate formatelyase is the main
decomposition pathway of pyruvate under anaerobic conditions, with formate and
acetylcoenzyme A (acetyl-CoA) as the products. On the other hand, the
phosphotransacetylase and acetate kinase catalyze the conversion of pyruvate via acetyl-
CoA and acetyl-phosphate to acetate with ATP generation, which is the main pathway for
acetate production in E. coli (Fig. 2). The deletions of pflA/pflB, ackA or pta genes
significantly reduced the yields of acetate (by 53.5, 77.6 and 52.1% , respectively) and
formate (by 100, 71 and 93.5%, respectively)14 and resulted in a decrease in ATP generation
under the oxygen-limited condition. Consequently, glycolysis was intensified to make up the
ATP limitation with significantly higher D-lactate production (lactate yield increased by 39.6,
25.2 and 39.4%, respectively, in the pflA/pflB, ackA and pta mutants), which was required for
the balancing of the redox states.

Effects of multiple gene deletions

To boost lactate generation even more, numerous genes were deleted in a cumulative
manner. Given that single-gene deletions with minimal or negative repercussions might have
different outcomes when these genes were deleted together, the effects of cumulative
deletions of all the potential genes were also investigated. Unlike the effects shown in single-
gene deletions of adhE, cumulative mutations of adhE and the pta gene, for example, could
promote lactic acid buildup via anaerobic fermentation.  In a complex media, however, an
additional mutation in the ppc gene in the pta mutant resulted in homofermentative
synthesis of D-lactate of 62.2 g L1. When N2 was employed to achieve strict anaerobic
conditions, lactate of 138 g L1 was produced with a yield of 99 percent and a productivity of
6.3 g L1 h1 by aerobic and anaerobic two-phase fed batch fermentation. Further
improvement of the fermentation process in ALS974 reduced the amount of succinate and
ethanol byproducts. 24 In a wild type E. coli, we combined the maximal deletions of the
ackA, pta, pps, pflB, dld, poxB, adhE, and frdA genes. 14 Cumulative mutations of ackA, pta,
pflB, dld, adhE, and frdA could assist boost lactate yield and decrease byproduct buildup,
while cumulative mutations of pps and poxB had no effect on these parameters. Strain
B0013-070, with the deletion of all eight genes, produced 125 g L−1 D-lactate with an overall
volumetric productivity of 3.32 g L−1 h−1 during the aerobic and oxygen-limited two-phase
fermentation without involving a complex medium, special chemicals and/or an inert gas.
The D-lactate optical purity was greater than 99.9%, although it did not involve the mutation
of mgsA. These results have revealed the genetic manipulations that improved D-lactate
production by E. coli.

Production of LA from other substrate with metabolic engineered E.coli

In order to produce LA commercially the cost of glucose is challenging to use it as a substrate

continuously. Due to this problem, it is required to improve the E.coli strain that can convert
other inexpensive substrates toward the lactic acid.Product from different carbon sources,
including glycerol, xylose and sucrose, which are present in byproducts from other industries
are optional substrates for production of lactic acid.

D-lactate production from sucrose

Sucrose is a major element of sugarcane and/or beet molasses and has been found as the
most cost-effective carbon source for industrial fermentation. However, only around half of
E. coli strains have the ability to ferment sucrose naturally. Cloning the sucrose gene cluster
(cscR, cscA, and cscKB, which encode a repressor protein, invertase, fructokinase, and anion
symport, respectively) from E. coli KO11 and over-expressing it in SZ63 (which lacks the
innate capacity to ferment sucrose) resulted in >45 g L1 D-lactate. 50 Furthermore, using
KO11 as a host strain, Zhou et al.19 created SZ132, a D-lactate generating strain that
produced >90 g L1 D-lactate in a complex medium containing 100 g L1 sucrose with a 95%
yield.Subsequently, the addition of betaine improved the D-lactate productivity and
tolerance to sugar, and eliminated the need for complex nutrients.20 In mineral salt media
supplemented with 1 mmol L−1 betaine, SZ132 produced 94 g L−1 D-lactate in 120 h (Table
1). However, the addition of betaine also decreased the chiral purity to 95%. Wang et al.
involved an additional gene deletion of aldA (encodes aldehyde dehydrogenase A that
generates L-lactate) in a sucrose-positive E. coli, and the D-lactate optical purity was
improved to 98.3% in strain HBUT-D51 .

D-Lactate production from xylose

Cellulosic biomass is the most abundant non-food resource. Lignocellulose hydrolyzes

contain significant amounts of xylose, which is the second most abundant monosaccharide
after glucose. However, xylose is rarely present as a monomer, and most fermenting
organisms are unable to utilize it as a carbon source. Therefore, highly efficient
bioconversion of xylose becomes one of the vital factors that can affect the industrial
prospects of lignocellulose applications. Some E. coli strains are able to metabolize xylose
under fermentative conditions to produce D-lactate with the use of mineral media.
Homolactic fermentation of pentoses in E. coli is redox balanced by reducing pyruvate into
D-lactate. However, the growth limitation is caused by an ATP deficit under the anaerobic
conditions. By deleting the ATP-dependent transporter XylFGH (xylose ABC transporter)
from the D-lactate producer E. coli CL3 (MG1655, pflBadhEfrdA), the growth rate was
increased by 50%. After an adaptive evolution to produce E. coli JU15, the D-lactate yield
reached 95% with a volumetric productivity of 0.79 g L−1 h−1 (Table 1). The high xylose
consumption rate (2.7 g g−1 h−1) was caused by the newly found xylose transporter GatC
(the mutation from serine to leucine at position 184 of the GatC protein caused higher
xylose transport ability), and it could be useful for converting C5–C6 syrups into valuable
chemicals.

D-lactate production from glycerol

A surplus of crude glycerol has been created as international biodiesel production has
increased. The associated economic and environmental disadvantages would be mitigated
by the efficient use of glycerol as a low-cost raw material. Furthermore, the synthesis of
optically pure D-lactate from raw glycerol using metabolically altered E. coli strains is
potentially viable, as the overall production costs in all cases were lower than the sale price
of D-lactate. Glycerol is a great prospective carbon source for producing reduced
compounds with high yields due to its inexpensive cost and abundance, as well as its higher
degree of reduction compared to sugars like glucose and xylose. With a yield of 85 percentof
the theoretical maximum, the modified homofermentative E. coli strain transformed 40 g L1
glycerol to 32 g L1 D-lactate. Chen et al., on the other hand, moderately increased D-lactate
production by improving D-LDH expression on a low copy number plasmid using strain
B0013-070-pTHldhA (B0013-070 strain38 with ldhA overexpression on plasmid pTH-ldhA)
and using the optimal cell growth and D-lactate fermentation temperatures, respectively.
With an overall productivity of 2.78 g L1 h1, a yield of 75.4 percent, and a minimum of by-
products, the D-Lactate titer was enhanced to 100.3 g L1. The highest degree of D-lactate
synthesis utilizing glycerol as the sole carbon source has been recorded.
 General metabolic pathway for the production of D-lactate from various substrates by E.
coli strains. Genes and enzymes: cscB sucrose permease (anion symport), cscA sucrose
hydrolase (invertase), cscK fructokinase, glk glucokinase, pts posphotransferase system
(PTS), pfkAB phosphofructokinase, fba fructose-1,6-bisphosphate aldolase, gatC galactitol-
specific enzyme IIC component of PTS, xylFGH xylose ABC transporter, glpK glycerol
kinase, glpD aerobic glycerol-3-phosphate dehydrogenase, gldA glycerol
dehydrogenase, dhaKLM dihydroxyacetone kinase, ldhA fermentative D-lactate
dehydrogenase. The abbreviated metabolic intermediate are: PEP phosphoenolpyruvate and
QH2 reduced quinines. The dotted arrow shows the same type of metabolic chemical. A
closed circle indicates that the gene was deleted.

8. . Purification of lactic acid

 9. Application of lactic acid (AbdAlsaheb, R. A., Aladdin, A., Othman, N. Z., AbdMalek, R., Leng,
O. M., Aziz, R., & El Enshasy, H. A. (2015). Lactic acid applications in pharmaceutical and
cosmeceutical industries. Journal of Chemical and Pharmaceutical Research, 7(10), 729-
735.)

In Food Inchemical In cosmetic In chemical In pharmaceutical

industry industry industry feedstock industry
-acidulates -descaling -moisturizers -propylene oxide -parenteral/I.V.
-preservatives agents -skin- -acetaldehyde solution
-flavoring -pH regulators lightening -acrylic acid -dialysis solution
agent -neutralizers agents -propionic acid -mineral preparations
-pH regulators -chiral -skin- -2,3-pentanedione -tablet tings
-improving intermediates rejuvenating -ethyl lactate -prostheses
microbial -green solvents agents -poly(lactic acid) -surgical sutures
quality -cleaning agents -pH regulators -controlled drug
-mineral -slow acid -anti-acne delivery system
fortification Releasing agents
agents -humectants
-Anti tartar
agents

Conclusion

Lactic acid is widely used chemical in the food, cosmetic, pharmaceutical, and chemical
industries and has received increased attention for use as a monomer for the production of
biodegradable poly(lactic acid). It can be produced by either biotechnological fermentation or
chemical synthesis. Homofermentative and hetrofermentative bacteria are the common bacteria
that are used in the production of lactic acid industrially. The high demand of lactic acid in
different application researcher work on high yields and quality of this product through
metabolic engineering of different hosts. Metabolic engineering of E. coli for the production of
lactic acid is very common and effective way for its suitability as compared to other hosts. Due to
its expensiveness of glucose as the substrate; production of lactic acid from glycerol, xylose and
sucrose are inexpensive by products that researcher looking for by engineering E.coli
metabolically. The further improvement of this strain can produce products in high volumetric
amount for the commercial and economical value.
 Why e.coli become dominant production host for different product
Historically, following the emergence of the biopharmaceutical industry in the 1980’s, E. coli
dominated as the main production organism used in >60% of cases for biopharmaceutical protein
production (Huang, Lin and Yan 2012). This was due in part to a combination of a short doubling
time, ability to grow to high cell densities and the relatively simple scale-up procedure.

Lactic acid production by recombinant E. coli BAD85

During the anaerobic growth of recombinant E. coli BADldh in fructose, lactic acid was produced
concurrently with cell growth (Figure 4). The concentration of lactic acid produced was achieved at
0.62 g l -1 with the productivity achieved at 0.026 g l -1 h -1 . The cell growth entered death phase
after fructose was depleted. At the end of fermentation, the final pH was reduced to pH 5.0 due the
acid accumulation. It was apparent that, as pointed out by Russel (1992) and Diez-Gonzalez and
Russel (1997) as the medium pH was farther away from the internal pH maintained by the bacteria,
inhibition effect increased regardless of whether the lactic acid was in the dissociated or
undissociated form. Tang et al. (1989) have shown that both dissociated and undissociated lactic
acid had inhibitory effects with the undissociated organic acids being more inhibitory than the
dissociated acids. According to Padan et al. (1981), cell maintained the intracellular pH by pumping H
+ out via a cationdependent proton pumps, which was an energy-intensive process. Meanwhile, the
anion concentration increased because the charge on the anion prevented it from transversing the
membrane (unless through a transport protein), which led to an osmotic imbalance (Russel, 1992;
Roc et al., 1998). Russel (1992) and Roc et al., (1998) further added that increasing the amount of
undissociated lactic acid, as occurred when lactic acid concentration was increased or the pH was
lowered, led to greater inhibition of cell growth because of pH and osmotic imbalances.

Escherichia coli, with the ability to utilize hexose and pentose sugars have been engineered for the
production of D-lactic acid [2–4, 12, 16–19, 21,27–29, 32, 33]. Nevertheless, most studies reported
the D-lactic acid production from glucose and/or sucrose by an engineered E. coli strain, with a titer,
productivity and yield of 80–120 g L−1, 2–6 g L−1h−1 and 80–95 %, respectively. Few of these strains,
however, have the ability to ferment xylose into D-lactic acid with a desired titer, yield and rate [3,
23, 24, 30, 31]. Furthermore, none, if any, of these strains have demonstrated the ability to co-
metabolize both glucose and xylose for enhanced D-lactic acid fermentation.

Like most bacteria, E. coli has a preference for glucose over other sugars for energy [7, 13].
Whenever glucose and other sugars are available, it will use glucose first, then other sugars only if
glucose is completely consumed. This phenomena is often called the glucose effect or catabolite
repression. The sequential use of glucose and xylose often results in delayed and incomplete use of
xylose for lactic acid fermentation using sugar mixtures [8, 23]. Eliminating the glucose effect is
needed to allow co-utilization of glucose and xylose for improved D-lactic acid production using
cellulosic substrates [11, 14].

In this study, we report reengineering E. coli WL204 (ΔfrdBCΔldhAΔackAΔpflBΔpdhR ::pflBp6-acEF-

lpd ΔmgsAΔadhE, ΔldhA::ldhL) [30] for D-lactic acid production by 1) replacing the L-lactate
dehydrogenase gene (ldhL) with a D-lactate dehydrogenase gene (ldhA); 2) eliminating catabolite
repression via deletion of the ptsG gene that encodes for IIBCglc, a major enzyme of the glucose PTS
system; 3) adaptive evolution in screw-cap tubes for improved cell growth with glucose as the sole
substrate. The resulting strain, E. coli JH15, is able to co-utilize both glucose and xylose for enhanced
D-lactic acid production[Lu, H., Zhao, X., Wang, Y., Ding, X., Wang, J., Garza, E., ...& Zhou, S. (2016).
Enhancement of D-lactic acid production from a mixed glucose and xylose substrate by the
Escherichia coli strain JH15 devoid of the glucose effect. BMC biotechnology, 16(1), 1-10.].