Identifying Bottlenecks in Engineering Efficient Cellobiose Metabolism

(Evidence for putative promoters within operon and TCA cycle imbalance)

Despite the availability of sophisticated genome editing tools, it is a seemingly difficult task to optimize the metabolic pathway and obtain mutants with desired functions. Often the failure to conquer the complexity of biological systems is because of the gaps in our understanding of the regulatory mechanisms governing the system under investigation. For example, even with a well- studied organism (i.e. Escherichia coli) about 50% of the regulatory mechanisms remain unknown. Indeed, it would take another decade to reduce the percentage of unknowns from 50% to 45%.
While the main purpose of such complex regulatory mechanisms is to choose the best carbon source that demands a minimal cellular resource for a maximal growth, metabolic engineers strive to engineer cells with deregulated metabolism for cellulose, a complex carbon source that requires the investment of a vast amount of cellular resources for cellulase production. The increase in demand for the production of value-added biochemicals from lignocelluloses urges to annihilate the regulatory networks (rather than to decipher them) to efficiently express several cellulases. Understanding the molecular mechanism of cellulose utilization in native cellulolytic organisms helped envision the fact that celluloses are metabolized in the form of cellodextrins and cellobioses (rather than as glucoses), in order to save the resources spent on secreting one of the enzymes, ?-glucosidase. Recently, E. coli is engineered for internal cellobiose metabolism using cryptic genes of E. coli.
In native E. coli, cellobiose metabolism is pertained to two cryptic operons: the chb operon and the asc operon. Since promoters of these two operons are relatively insensitive to cellobiose,
wild-type E. coli is unable to grow on cellobiose as a sole carbon source. We replaced the native promoters of these two operons with synthetic constitutive promoters and the resulting strain (SVC01) acquired the ability to grow on cellobiose. But the growth rate of SVC01 was very low (µ= 0.21/hr). Adaptive evolution on cellobiose for 30 days helped in enhancing the growth rate of SVC01 (µ= 0.40/hr).
Even when E. coli is engineered to metabolize cellobiose efficiently, their cellobiose metabolic rate was lower than the glucose metabolic rate. In addition, these strains suffered from repression of cellobiose metabolism in the presence of glucose thus failing to mimic the native cellulolytic organisms. Here, we identified two major bottlenecks in efficient cellobiose metabolism: 1) regulation of the internal putative promoters of asc operon and 2) difference in the pathway for the metabolism of glucose and cellobiose. We show that it is possible to rewire the regulations of the cryptic genes of E. coli in order to increase the rate of cellobiose metabolism.

Uncharacterized putative promoters within asc operon:

Use of synthetic constitutive promoters or modularized assembly of genes as an operon became a potent alternative to circumvent the host-dependent regulations such as stationary phase effect, sigma-factor dependence or carbon catabolite repression of the desired pathway. Here, we show with cellobiose metabolism as a proof-of-concept that despite the use of synthetic promoters or operons, the presence of putative, uncharacterized promoters within an operon is another bottleneck in engineering the metabolic pathways. The mutations responsible for the enhanced growth rate of SVC01 were biased around the RBS of ascB gene of the asc operon. Optimization of the RBS of all genes of the two operons and their promoters through single stranded oligo- mediated recombineering also enhanced the growth rate (µ= 0.32/hr) of SVC01. The asc operon
encodes two genes where ascF, the first gene of the operon, encodes the incomplete cellobiose transporter and the second gene, ascB encodes the enzyme ?-phosphoglucosidase. Analysis of the mRNA level between SVC01 and the adapted strain indicates no change in the expression of the first gene, ascF but a 5-fold increase in the expression of the second gene, ascB. The differential expression of the first and the second gene intrigues the possibility of an internal promoter in this operon. A promoter region was identified between -70bp and -570bp from the start codon of ascB gene. The regulations of this putative promoter were being deciphered in order to enhance the growth rate of SVC01 on cellobiose.

Difference in the routes for metabolism of glucose and cellobiose

We hypothesized that the cellobiose metabolic rate could be enhanced, if cellobiose is metabolized through the same pathway as that of glucose. In order to identify the pathway difference between cellobiose and glucose, the total proteome of cells grown on these carbon sources were compared. Interestingly, the expression of glyoxylate pathway enzyme was very high whereas the expression of ED pathway enzyme was reduced in cellobiose grown cells compared to cells growing on glucose minimal medium. The expression level of glyoxylate pathway protein, AceB was followed by western blotting. As expected, the glyoxylate pathway in the adapted strains was constitutively expressed at a higher level in cells growing on cellobiose-minimal medium when compared to cells grown on glucose-minimal medium.
This result indicates an imbalanced distribution of flux around the glyoxylate pathway and the ED pathway in cellobiose when compared to glucose metabolism. Rerouting the flux of cellobiose metabolism to mimic the glucose metabolic pathway might be one way to optimize the metabolic pathway. Hence, the flux distributions between these two pathways were altered
through the modulation of the promoter strength of the genes encoding these two pathways. Thus, the putative promoter in the asc operon and imbalanced flux distribution between ED pathway and the glyoxylate pathway are two major bottlenecks controlling efficient cellobiose metabolism in E. coli. In this study, we highlight that the putative promoters within an operon could be an important factor to be considered in metabolic engineering practices.

Fig 1: Remodeling cryptic operons: Remove internal promoters and regulatory regions

chbB chbA chbC chbF chbR chbG

ascF ascB

Usual targets optimized: Op eron p romoters and RBS

New targets to be optimized: Internal p romotes

Fig 2: Comparison of glucose and cellobiose metabolism

G6P

pgl

6PGC

gnd

E4P

pfkA

pfkB

F6P

F1,6P GAP

KDPG

eda

PYR

ACA

OAA

GLO

aceA

CIT

icd

Expressed only in Glucose High in Cellobiose Expressed in both

SUC

AKG

SUCOA