Classical strain improvement

It is not always possible to obtain a strain with the exact combination of properties required for a specific use by screening of natural isolates. Thus, it is often necessary 

These treatments are relatively nonspecific and generate mutations at random in the genome. Cells with the desired properties are subsequently obtained from this population by screening of a large number of isolates or selection of variants with the desired property. 

The tools developed for soeening of natural isolates (Section 10.2.3) can also be used for screening of the survivors of a mutagenesis procedure for strains with desired properties. This will be illustrated with a few examples from our own laboratories. 

Lactobacillus plantarum is used in the wine industry, where it performs malo-lactic fermentation following the alcoholic fermentation by the wine yeast (Lerm, Engelbrecht, du Toit, 2011). This fermentation is important for the organoleptic properties of the wine because the relatively harsh malic acid is converted to the 

The bacterial surface is also involved in the formation of texture in fermented dairy prodncts such as yogurt. Consumers today prefer products that have a mild flavor and a high level of texture. Mutants of S. thermophilus and L. delbrueckii snbsp. bulgaricus resistant to D-cycloserine were selected and tested for their ability to grow in milk. It was observed that several of the mutants produce more texture and less whey than the parent strain and combining mutants of each species produced yogurt with properties satisfying the current consumer preference (Kibenich, Sprensen, Johansen, 2012). 

---

Classical strain improvement can be used, for example, to address the regulation of an amino acid by selecting mutants that are able to grow in the presence of a mimic that has similar regulation characteristics. The classical approach involves random mutagenesis and addresses the whole genome. Hence, the phenotype is primarily targeted and the actual mechanism is not easily subjected to rational control. 

In the cases described below we will generally focus on metabolic pathway engineering rather than on classical strain improvement, for obvious reasons. One should be aware, however, that the classical approach has strengths that can make it a powerful partner of the rational approach. Thus, regulation problems have been addressed by the development of a feedback-resistant enzyme, using selective pressure and random mutagenesis as described above, in a research species, followed by introduction of the altered gene in the production species via recombinant techniques. 

This intractable problem may now be close to being solved. A Saccharomyces species that expressed the xylose isomerase gene from an anaerobic fungus was found to grow slowly on pentoses [29]. Improvement resulted from a combination of rational engineering - overexpression of the pentose phosphate-converting enzymes (see Fig. 8.5) - and classical strain improvement [30]. The authors conclude The kinetics of xylose fermentation are no longer a bottleneck in the industrial production of ethanol with yeast ... 

In the early years, L-Phe was microbially produced with Corynebacterium gluta-micum and E. coli strains which had been deregulated with respect to the end product via classical strain improvement. More recently, metabolic engineering has been employed to address nearly all aspects of the biosynthesis of L-Phe the work has been reviewed [70, 93]. 

Little is known about the current production levels of L-Phe, but in the early days of metabolic engineering 50 g L-1 of L-Phe, with a yield on glucose of 0.27 mol mol-1, was produced by an engineered E. coli [92]. This is near the theoretical limit [91] and one would surmise that in the starting species used by these authors some PEP-conserving mutations had been introduced via classical strain improvement. 

One of the drawbacks in the current commercial fermentation process is that the predominant form of the product is the deprotonated lactate rather than lactic acid, requiring more expensive and wasteful product purification steps. This is because the Lactobacillus fermentation operates at a minimum pH of 5.0-5.5 which is above the pA a of lactic acid (3.87). To overcome this limitation, a powerful strain improvement method, genome shuffling, was used to improve the acid tolerance of a poorly characterized industrial strain of LactobacillusA population of strains with subtle improvement in pH tolerance was isolated using classical strain improvement methods such as chemostats, and were then shuffled by recursive pool-wise protoplast fusion to create mutant strains that grow at substantially lower pH than does the wild-type strain. 

Product yield is very often a factor that limits the development of a natural product as a lead compound. Traditionally, both chemical synthesis and classical strain improvement technologies have been applied to overcome this limitation. As natural products are often molecules of high structural and stereochemical complexity, their total synthesis is usually difficult, and yields are low. Classical strain improvement represents an equally time-consuming and rather undirected process, during which numerous rounds of mutagenesis and subsequent screening are applied to obtain strains with improved production titers. 

Classical Strain Improvement and Whole Genome Sequencing 131... 

Lai R, Khanna R, Kaur H, Khanna M, Dhingra N, Lai S, Gartemann K H, Eichenlaub R, Ghosh P K (1996). Engineering antibiotic producers to overcome the limitations of classical strain improvement programs. Crit. Rev. Microbiol. 22 201-255. 

Polyketide natmal products are a rich source of bioactive substances that have found considerable use in hrnnan health and agriculture. Their complex structures require that they be produced via fermentation processes. This review describes the strategies and challenges used to develop practical fermentation strains and processes for polyketide production. Classical strain improvement procedmes, process development methods, and metabolic engineering approaches are described. The elucidation of molecular mechanisms that imderlie polyketide biosynthesis has played an important role in each of these areas over the past few years. 

Significance of Classical Strain Improvement in Times of Synthetic Biology 247... 

A standardized work flow for strain acquisition, strain purification, primary characterization, and where necessary, classical strain improvement was initiated in our laboratories in 2(X)8 and designated the strain supply chain (SSC). The introduction of these four standardized systematic work processes has resulted in a faster and more resource-efficient development of new strains, both wild-type strains and classically improved strains, for new product development. It is presented here to serve as an inspiration for other groups seeking to systematize their strain-screening activities (Figure 10.1). 

Crook, N., Alper, H. (2013). Classical strain improvement. In R. Patnaik (Ed.), Engineering complex phenotypes in industrial strains (pp. 1-33). Hoboken John Wiley Sons, Inc. 

---